Bacterial and viral infections represent some of the most difficult diseases to treat in humans due to resistance to most of the therapeutic agents. The emergence of drug resistance factors that have threatened the efficacy of all antibacterial agents prompted the investigation of antimicrobial activity studies of methanol extracts from Durio zibethinus Murr. This study is focused on evaluating the phytochemical properties, antibacterial efficacy of methanolic root extract of D. zibethinus Murr. against gram-positive bacterial strains (Staphylococcus aureus and Staphylococcus epidermidis) and gram-negative bacterial strains (Klebsiella pneumonia, Escherichia coli and Salmonella typhi). Phytochemical properties of methanolic root extract of D. zibethinus Murr were investigated using qualitative analysis, while the antibacterial was evaluated using standard agar disc diffusion technique. The results revealed the presence of alkaloids, saponins, tannins, flavonoids, terpenoids, steroids, glycosides, and phenolic compounds in the D. zibethinus Murr. root. It also indicated that the root extract exhibited antibacterial activity against S. aureus, S. epidermidis, K. pneumonia and S. typhi at a range of 12.07 – 24.01 mm at the highest concentration of 150 mg/mL relative to standard gentamicin antibiotic. The minimum inhibitory concentration (MIC) for S. aureus was 0.250 mg/mL, whereas it was 0.125 mg/mL for S. epidermidis, K. pneumonia and S. typhi. In the present study, root extract of D. zibethinus Murr. showed the highest antibacterial activity against S. aureus. Hence, D. zibethinus Murr. root can be used as new source for antibacterial substance.